1. Field of the Invention
The present invention relates to a method of assay of activity of an enzyme employed as a label for immunoassay, etc. based on the detection of fluorescence intensity of a substance having been affected by the enzyme. The present invention also relates to an apparatus used for the assay of the activity of the enzyme.
2. Description of the Related Art
Conventionally known methods for assaying a minute quantity of a biological substance include labeled immunoassay and enzyme-labeled DNA hybridization.
These methods are based on a principle that the biological substance is identified or determined by assaying the activity of an enzyme linked directly or indirectly to an immune complex formed through an immune reaction, or the like, according to detection of the change of the substrate. The known methods include measurement of the change of a light absorption coefficient, measurement of the change of fluorescence intensity, measurement of the change of light emission intensity, of a sample having been affected by enzyme activity, and so forth. From among these methods, the last two methods including measurement of enzyme activity by fluorescence or light emission are advantageous in that a higher sensitivity is achievable in shorter time than in the method which utilizes the light absorption for enzymatic activity assay. The fluorescence method is known to be highly sensitive for assaying enzymatic activity.
Several methods have been reported for quantitative determination of biological substances on the basis of change of fluorescence intensity of a sample. The examples are a method in which the measurement is conducted at a specific point of time after the initiation of an enzymatic reaction and the enzymatic activity is derived from the measured data (a one-point method); a method in which the measurement is conducted at two points of time during the progress of a series of enzymatic reactions and the enzymatic activity is derived from the difference of the two measured data (a two-point method); and a method in which the measurement is conducted nearly continuously and the enzymatic activity was derived from the change rate of the continuously measured data (a rate method).
In recent years, the significance and the importance of quantitative determination of a minute quantity of a substance have been given more attention, in particular, in the fields such as clinical diagnosis. Consequently, the determination of a minute quantity of a biological sample with high precision and high sensitivity has been increasingly required. To meet such requirement, proposals have been made regarding a system which is free from contamination of a sample by another sample, a detecting device for successive measurement of a plurality of samples with high sensitivity, and so forth.
The methods and apparatus of prior arts, which have been proposed or been put into commercial practice for quantitative determination of biological substances with high sensitivity and high precision, have been confronted with limitation in improvement of precision of measurement more than a certain degree because of significant influence of air bubbles in the sample and meniscus of the sample liquid, and so forth. One reason for the above limitation in the improvement is that samples of biological substances to be measured are available only in a minute amount in most cases.
For example, the presence of bubbles in the sample liquid gives a larger value of fluorescence measurement than a value measured in the absence of the bubbles, so that disappearance of the bubbles during the measurement will give, for example, a lower value in a rate method than the real value. On the other hand, the injection of the substrate, which is to be subjected to the reaction with a label enzyme, into a reaction vessel is required to be conducted as quickly as possible in order to treat quickly a number of samples. Further, any adhesion of a liquid drop at the tip of the nozzle needs to be avoided at the completion of dispensing of the sample liquid in order to obtain high accuracy of the volume to be dispensed. However, if the liquid ejection speed is made higher to satisfy the above requirements, the dispensed liquid tends to be foamed more. In the enzyme-labeled immunoassay, an immune reaction complex and an unreacted matter are generally separated from each other (B/F separation) before the substrate injection. If the concentration of surface active agent, which is usually contained in the washing solution, is made higher, more bubbles tend to be formed.
Accordingly, more and more demands have been made for comprehensive investigations to be made to find a novel measurement method which is free from the above problems in principle and enables improvement of the accuracy of the measurement.
Apart from the above problems, a higher detection sensitivity is required in fluorescence measurement. For example, in immunoassay of TSH (thyroid-stimulating hormone), the lower detection limit has conventionally been at a level of about 0.1 .mu.IU/ml, but the limit is now required to be at a level of 0.01 .mu.IU/ml for diagnosis of disease state of an abnormal low value of TSH. Therefore, a higher detection sensitivity is required as well as the suppression of nonspecific reaction of the labeled enzyme. For the improvement of the detection sensitivity, the change of the real measured value has to be distinguished from the variation caused by external disturbance.